Dogs develop complex multifactorial diseases analogous to humans, including inflammatory diseases, metabolic diseases, and cancer. Therefore, they represent relevant large animal models with the translational potential to human medicine.Organoids are 3-dimensional (3D), self-assembled structures derived from stem cells that mimic the microanatomy and physiology of their organ of origin. These translational in vitro models can be used for drug permeability and discovery applications, toxicology assessment, and to provide a mechanistic understanding of the pathophysiology of multifactorial chronic diseases. Furthermore, canine organoids can enhance the lives of companion dogs, providing input in various areas of veterinary research and facilitating personalized treatment applications in veterinary medicine. A small group of donors can create a biobank of organoid samples, reducing the need for continuous tissue harvesting, as organoid cell lines can be sub-cultured indefinitely.Herein, three protocols that focus on the culture of intestinal and hepatic canine organoids derived from adult stem cells are presented. The Canine Organoid Isolation Protocol outlines methods to process tissue and embedding of the cell isolate in a supportive matrix (solubilized extracellular membrane matrix). The Canine Organoid Maintenance Protocol describes organoid growth and maintenance, including cleaning and passaging along with appropriate timing for expansion. The Organoid Harvesting and Biobanking Protocol describes ways to extract, freeze, and preserve organoids for further analysis.
Dogs develop complex multifactorial diseases analogous to humans, including inflammatory diseases, metabolic diseases, and cancer. Therefore, they represent relevant large animal models with the translational potential to human medicine.Organoids are 3-dimensional (3D), self-assembled structures derived from stem cells that mimic the microanatomy and physiology of their organ of origin. These translational in vitro models can be used for drug permeability and discovery applications, toxicology assessment, and to provide a mechanistic understanding of the pathophysiology of multifactorial chronic diseases. Furthermore, canine organoids can enhance the lives of companion dogs, providing input in various areas of veterinary research and facilitating personalized treatment applications in veterinary medicine. A small group of donors can create a biobank of organoid samples, reducing the need for continuous tissue harvesting, as organoid cell lines can be sub-cultured indefinitely.Herein, three protocols that focus on the culture of intestinal and hepatic canine organoids derived from adult stem cells are presented. The Canine Organoid Isolation Protocol outlines methods to process tissue and embedding of the cell isolate in a supportive matrix (solubilized extracellular membrane matrix). The Canine Organoid Maintenance Protocol describes organoid growth and maintenance, including cleaning and passaging along with appropriate timing for expansion. The Organoid Harvesting and Biobanking Protocol describes ways to extract, freeze, and preserve organoids for further analysis.
The permeable support system is typically used in conjunction with traditional twodimensional (2D) cell lines as an in vitro tool for evaluating the oral permeability of new therapeutic drug candidates. However, the use of these conventional cell lines has limitations, such as altered expression of tight junctions, partial cell differentiation, and the absence of key nuclear receptors. Despite these shortcomings, the Caco-2 and MDCK models are widely accepted and validated for the prediction of human in vivo oral permeability. Dogs are a relevant translational model for biomedical research due to their similarities in gastrointestinal anatomy and intestinal microflora with humans. Accordingly, and in support of parallel drug development, the elaboration of an efficient and accurate in vitro tool for predicting in vivo drug permeability characteristics both in dogs and humans is highly desirable. Such a tool could be the canine intestinal organoid system, characterized by three-dimensional (3D), self-assembled epithelial structures derived from adult stem cells.The (1) Permeable Support Seeding Protocol describes the experimental methods for dissociating and seeding canine organoids in the inserts. Canine organoid isolation, culture, and harvest have been previously described in a separate set of protocols in this special issue. Methods for general upkeep of the canine intestinal organoid monolayer are discussed thoroughly in the (2) Monolayer Maintenance Protocol.Additionally, this protocol describes methods to assess the structural integrity of the monolayer via transepithelial electrical resistance (TEER) measurements and light microscopy. Finally, the (3) Permeability Experimental Protocol describes the tasks directly preceding an experiment, including in vitro validation of experimental results.
Chronic inflammatory enteropathy (CE) is a common cause of persistent gastrointestinal signs and intestinal inflammation in dogs. Since evidence links dysbiosis to mucosal inflammation, probiotics, prebiotics, or their combination (synbiotics) may reduce intestinal inflammation and ameliorate dysbiosis in affected dogs. This study’s aim was to investigate the effects of the synbiotic-IgY supplement on clinical signs, inflammatory indices, and mucosal microbiota in dogs with CE. Dogs with CE were enrolled in a randomized prospective trial. Twenty-four client-owned dogs were fed a hydrolyzed diet and administered supplement or placebo (diet) for 6 weeks. Dogs were evaluated at diagnosis and 2- and 6-week post-treatment. Outcome measures included clinical activity, endoscopic and histologic scores, inflammatory markers (fecal calprotectin, C-reactive protein), and composition of the mucosal microbiota via FISH. Eleven supplement- and nine placebo-treated dogs completed the trial. After 6 weeks of therapy, clinical activity and endoscopic scores decreased in both groups. Compared to placebo-treated dogs, dogs administered supplement showed decreased calprotectin at 2-week post-treatment, decreased CRP at 2- and 6-week post-treatment increased mucosal Clostridia and Bacteroides and decreased Enterobacteriaceae in colonic biopsies at trial completion. Results suggest a beneficial effect of diet and supplements on host responses and mucosal microbiota in dogs with CE.
A key component of efforts to identify the biological and drug-specific aspects contributing to therapeutic failure or unexpected exposure-associated toxicity is the study of drug–intestinal barrier interactions. While methods supporting such assessments are widely described for human therapeutics, relatively little information is available for similar evaluations in support of veterinary pharmaceuticals. There is, therefore, a critical need to develop novel approaches for evaluating drug–gut interactions in veterinary medicine. Three-dimensional (3D) organoids can address these difficulties in a reasonably affordable system that circumvents the need for more invasive in vivo assays in live animals. However, a first step in developing such systems is understanding organoid interactions in a 2D monolayer. Given the importance of orally administered medications for meeting the therapeutic need of companion animals, we demonstrate growth conditions under which canine-colonoid-derived intestinal epithelial cells survive, mature, and differentiate into confluent cell systems with high monolayer integrity. We further examine the applicability of this canine-colonoid-derived 2D model to assess the permeability of three structurally diverse, passively absorbed β-blockers (e.g., propranolol, metoprolol, and atenolol). Both the absorptive and secretive apparent permeability (Papp) of these drugs at two different pH conditions were evaluated in canine-colonoid-derived monolayers and compared with that of Caco-2 cells. This proof-of-concept study provides promising preliminary results with regard to the utility of canine-derived organoid monolayers for species-specific assessments of therapeutic drug passive permeability.
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